The present invention relates to a method for producing glucosamine by fermentation. The present invention also relates to genetically modified strains of microorganisms useful for producing glucosamine.
Amino sugars are usually found as monomer residues in complex oligosaccharides and polysaccharides. Glucosamine is an amino derivative of the simple sugar, glucose. Glucosamine and other amino sugars are important constituents of many natural polysaccharides. For example, polysaccharides containing amino sugars can form structural materials for cells, analogous to structural proteins.
Glucosamine is manufactured as a nutraceutical product with applications in the treatment of osteoarthritic conditions in animals and humans. The market for glucosamine is experiencing tremendous growth. Furthermore, significant erosion of the world market price for glucosamine is not expected.
Glucosamine is currently obtained by acid hydrolysis of chitin, a complex carbohydrate derived from N-acetyl-D-glucosamine. Alternatively, glucosamine can also be produced by acid hydrolysis of variously acetylated chitosans. These processes suffer from poor product yields (in the range of 50% conversion of substrate to glucosamine). Moreover, the availability of raw material (i.e., a source of chitin, such as crab shells) is becoming increasingly limited. Therefore, there is a need in the industry for a cost-effective method for producing high yields of glucosamine for commercial sale and use.
One embodiment of the present invention relates to a method to produce glucosamine by fermentation of a microorganism. This method includes the steps of: (a) culturing in a fermentation medium a microorganism having a genetic modification in an amino sugar metabolic pathway; and (b) recovering a product produced from the step of culturing which is selected from the group of glucosamine-6-phosphate and glucosamine. Such an amino sugar metabolic pathway is selected from the group of a pathway for converting glucosamine-6-phosphate into another compound, a pathway for synthesizing glucosamine-6-phosphate, a pathway for transport of glucosamine or glucosamine-6-phosphate out of the microorganism, a pathway for transport of glucosamine into the microorganism, and a pathway which competes for substrates involved in the production of glucosamine-6-phosphate. The fermentation medium includes assimilable sources of carbon, nitrogen and phosphate. In a preferred embodiment, the microorganism is a bacterium or a yeast, and more preferably, a bacterium of the genus Escherichia, and even more preferably, Escherichia coli. 
In one embodiment, the product can be recovered by recovering intracellular glucosamine-6-phosphate from the microorganism and/or recovering extracellular glucosamine from the fermentation medium. In further embodiments, the step of recovering can include purifying glucosamine from the fermentation medium, isolating glucosamine-6-phosphate from the microorganism, and/or dephosphorylating the glucosamine-6-phosphate to produce glucosamine. In one embodiment, at least about 1 g/L of product is recovered.
In yet another embodiment, the step of culturing includes the step of maintaining the carbon source at a concentration of from about 0.5% to about 5% in the fermentation medium. In another embodiment, the step of culturing is performed at a temperature of from about 28xc2x0 C. to about 37xc2x0 C. In yet another embodiment, the step of culturing is performed in a fermenter.
In one embodiment of the present invention, the microorganism has a modification in a gene which encodes a protein including, but not limited to, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetyl-glucosamine-specific enzyme IINag, glucosamine-6-phosphate synthase, phosphoglucosamine mutase, glucosamine-1-phosphate acetyltransferase-N-acetylglucosamine-1-phosphate uridyltransferase, phosphofructokinase, enzyme IIGlc of the PEP:glucose PTS, EIIM, P/IIIMan of the PEP:mannose PTS, and/or a phosphatase.
In another embodiment, the genetic modification includes a genetic modification which increases the action of glucosamine-6-phosphate synthase in the microorganism. Such a genetic modification includes the transformation of the microorganism with a recombinant nucleic acid molecule encoding glucosamine-6-phosphate synthase to increase the action of glucosamine-6-phosphate synthase and/or to overexpress the glucosamine-6-phosphate synthase by the microorganism. In one embodiment, the recombinant nucleic acid molecule is operatively linked to a transcription control sequence. In a further embodiment, the recombinant nucleic acid molecule is integrated into the genome of the microorganism. In yet another embodiment, the recombinant nucleic acid molecule encoding glucosamine-6-phosphate synthase has a genetic modification which increases the action of the synthase. Such genetic modifications can result in reduced glucosamine-6-phosphate product inhibition of the glucosamine-6-phosphate synthase, for example.
In one embodiment, a recombinant nucleic acid molecule of the present invention which comprises a nucleic acid sequence encoding a glucosamine-6-phosphate synthase encodes an amino acid sequence SEQ ID NO:16. In another embodiment, such a recombinant nucleic acid molecule comprises a nucleic acid sequence selected from the group of SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15. Preferred recombinant nucleic acid molecules of the present invention include pKLN23-28, nglmS-282184 and nglmS-281830.
Also included in the present invention are recombinant nucleic acid molecules encoding a glucosamine-6-phosphate synthase which comprises a genetic modification which increases the action of the glucosamine-6-phosphate synthase (i.e., a glucosamine-6-phosphate synthase homologue). Such a genetic modification can reduce glucosamine-6-phosphate product inhibition of the synthase, for example. In one embodiment, such a genetic modification in a recombinant nucleic acid molecule of the present invention which encodes a glucosamine-6-phosphate synthase results in at least one amino acid modification selected from the group of an addition, substitution, deletion, and/or derivatization of an amino acid residue of the glucosamine-6-phosphate synthase. In one embodiment, such an amino acid modification is in an amino acid sequence position in the modified protein (i.e., homologue) which corresponds to one or more of the following amino acid positions in amino acid sequence SEQ ID NO:16: Ile(4), Ile(272), Ser(450), Ala(39), Arg(250), Gly(472), Leu(469). In another embodiment, such an amino acid modification is selected from the group of a substitution of: (a) an amino acid residue having an aliphatic hydroxyl side group for Ile(4); (b) an amino acid residue having an aliphatic hydroxyl side group for Ile(272); (c) an amino acid residue having an aliphatic side group for Ser(450); (d) an amino acid residue having an aliphatic hydroxyl side group for Ala(39); (e) an amino acid residue having a sulfur-containing side group for Arg(250); (f) an amino acid residue having an aliphatic hydroxyl side group for Gly(472); (g) an amino acid residue having an aliphatic side group for Leu(469); and, (h) combinations of (a)-(g).
In yet another embodiment of the present invention, an amino acid modification as described above is preferably a substitution selected from the group of: Ile(4) to Thr, Ile(272) to Thr, Ser(450) to Pro, Ala(39) to Thr, Arg(250) to Cys, Gly(472) to Ser, Leu(469) to Pro, and combinations thereof.
In another embodiment, a genetically modified recombinant nucleic acid molecule of the present invention comprises a nucleic acid sequence encoding glucosamine-6-phosphate synthase comprising an amino acid sequence selected from the group of SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28 or SEQ ID NO:31. In another embodiment, a genetically modified recombinant nucleic acid molecule of the present invention comprises a nucleic acid sequence selected from the group of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29 and SEQ ID NO:30. Preferred genetically modified recombinant nucleic acid molecule of the present invention include pKLN23-49, pKLN23-54, pKLN23-124, pKLN23-149, pKLN23-151, nglmS-492184, nglmS-491830, nglmS-542184, nglmS-541830, nglmS-1242184, nglmS-1241830, nglmS-1492184, nglmS-1491830, nglmS-1512184 and nglmS-1511830.
Another embodiment of the present invention relates to a glucosamine-6-phosphate synthase which has glucosamine-6-phosphate synthase action, such synthase being encoded by a nucleic acid sequence having a genetic modification that results in increased glucosamine-6-phosphate synthase action. Such a nucleic acid sequence has been describe above with regard to recombinant nucleic acid molecules of the present invention.
Yet another embodiment of the present invention relates to a method to produce glucosamine by fermentation, such method comprising: (a) culturing in a fermentation medium comprising assimilable sources of carbon, nitrogen and phosphate, a genetically modified microorganism having increased glucosamine-6-phosphate synthase action, wherein the genetically modified microorganism is produced by a process comprising the steps of: 1) generating modifications in an isolated nucleic acid molecule comprising a nucleic acid sequence encoding glucosamine-6-phosphate synthase to create a plurality of modified nucleic acid sequences; (2) transforming microorganisms with the modified nucleic acid sequences to produce genetically modified microorganisms; (3) screening the genetically modified microorganisms for glucosamine-6-phosphate synthase action; and, (4) selecting the genetically modified microorganisms which have increased glucosamine-6-phosphate synthase action; and, (b) recovering the product. The step of culturing produces a product selected from the group of glucosamine-6-phosphate and glucosamine from the microorganism.
In another embodiment, a microorganism of the present invention has an additional genetic modification in genes encoding N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetyl-glucosamine-specific enzyme IINag, phosphoglucosamine mutase, glucosamine-1-phosphate acetyltransferase-N-acetylglucosamine-1-phosphate uridyltransferase, phosphofructokinase, Enzyme IIGlc of the PEP:glucose PTS, EIIM,P/IIIMan of the PEP:mannose PTS, wherein the modification decreases the action of such proteins. In another embodiment, a microorganism of the present invention has an additional genetic modification in a gene encoding a phosphatase, wherein the modification increases the action of the phosphatase. In a preferred embodiment, a microorganism of the present invention has an additional genetic modification in the genes encoding the following proteins: N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase and N-acetyl-glucosamine-specific enzyme IINag, such modifications including, but not limited to, a deletion of at least a portion of such genes.
Another embodiment of the present invention relates to a method to produce glucosamine by fermentation which includes the steps of (a) culturing an Escherichia coli transformed with a recombinant nucleic acid molecule encoding glucosamine-6-phosphate synthase in a fermentation medium comprising assimilable sources of carbon, nitrogen and phosphate to produce a product, and (b) recovering the product. The product includes intracellular glucosamine-6-phosphate which is recovered from the Escherichia coli and/or extracellular glucosamine which is recovered from the fermentation medium. In this embodiment, the recombinant nucleic acid molecule increases expression of the glucosamine-6-phosphate synthase by the Escherichia coli, and is operatively linked to a transcription control sequence. In one embodiment, the recombinant nucleic acid molecule comprises a genetic modification which reduces glucosamine-6-phosphate product inhibition of the glucosamine-6-phosphate synthase. In another embodiment, the Escherichia coli has an additional genetic modification in at least one gene selected from the group of nagqA nagB, nagC, nagD, nagE, manXYZ, glmM, pfkB, pfkA, glmU, glmS, ptsG and/or a phogphatage gene. In yet another embodiment, the additional modification comprises a deletion of nagA, nagB, nagC, nagD, nagE, and a mutation in manXYZ, wherein the modification results in decreased enzymatic activity of N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase and N-acetyl-glucosamine-specific enzyme IINag.
Yet another embodiment of the present invention relates to a microorganism for producing glucosamine by a biosynthetic process. The microorganism is transformed with a recombinant nucleic acid molecule encoding glucosamine-6-phosphate synthase, wherein the recombinant nucleic acid molecule is operatively linked to a transcription control sequence. The recombinant nucleic acid molecule further comprises a genetic modification which increases the action of the glucosamine-6-phosphate synthase. The expression of the recombinant nucleic acid molecule increases production of the glucosamine by the microorganism. In a preferred embodiment, the recombinant nucleic acid molecule is integrated into the genome of the microorganism. In yet another embodiment, the microorganism has at least one additional genetic modification in a gene encoding a protein selected from the group consisting of N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetyl-glucosamine-specific enzyme IINag, phosphoglucosamine mutase, glucosamine-1-phosphate acetyltransferase-N-acetylglucosamine-1-phosphate uridyltransferase, phosphofructokinase, Enzyme IIGlc of the PEP:glucose PTS, and/or EIIM,P/IIIMan of the PEP:mannose PTS, wherein the genetic modification decreases the action of the protein. In another embodiment, the microorganism has a modification in a gene encoding a phosphatase, wherein the genetic modification increases the action of the phosphatase. In yet another embodiment, the microorganism has a modification in genes encoding N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase and N-acetylglucosamine-specific enzyme IINag, wherein the genetic modification decreases enzymatic activity of the protein. In a preferred embodiment, the genetic modification is a deletion of at least a portion of the genes.
In a further embodiment, the microorganism is Escherichia coli, having a modification in a gene selected from the group of nagA, nagB, nagC, nagD, nagE, manXYZ, glmM, pfkB, pfkA, glmU, ptsG and/or a phosphatase gene. In one embodiment, such an Escherichia coli has a deletion of nag regulon genes, and in another embodiment, such an Escherichia coli has a deletion of nag regulon genes and a genetic modification in manXYZ genes such that the proteins encoded by the manXYZ genes have decreased action.
Yet another embodiment of the present invention is a microorganism as described above which produces at least about 1 g/L of glucosamine when cultured for about 10-60 hours at from about 28xc2x0 C. to about 37xc2x0 C. to a cell density of at least about 8 g/L by dry cell weight, in a pH 7.0 fermentation medium comprising: 14 g/L K2HPO4, 16 g/L KH2PO4, 1 g/L Na3Citrate.2H2O, 5 g/L (NH4)2SO4, 20 g/L glucose, 10 mM MgSO4, 1 mM CaCl2, and from about 0.2 mM to about 1 mM IPTG.
Another embodiment of the present invention is a microorganism for producing glucosamine by a biosynthetic process, which includes: (a) a recombinant nucleic acid molecule encoding glucosamine-6-phosphate synthase operatively linked to a transcription control sequence; and, (b) at least one genetic modification in a gene encoding a protein selected from the group of N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetyl-glucosamine-specific enzyme IINag, phosphoglucosamine mutase, glucosamine-1-phosphate acetyltransferase-N-acetylglucosamine-1-phosphate uridyltransferase, phosphofructokinase, Enzyme IIGlc of the PEP:glucose PTS, and/or EIIM,P/IIIMan of the PEP:mannose PTS, wherein the genetic modification decreases the action of the protein. In another embodiment, the microorganism includes at least one genetic modification in a gene encoding a phosphatase, wherein the genetic modification increases the action of the phosphatase. Expression of the recombinant nucleic acid molecule increases the action of the glucosamine-6-phosphate synthase in the microorganism. In a further embodiment, the recombinant nucleic acid molecule is integrated into the genome of the microorganism.